Amylase is an enzyme present in body fluids such as saliva, pancreatic juice, urine, serum and so on. Amylase activity measurement is an important item in the field of a clinical examination for a diagnosis of pancreas diseases such as chronic or acute pancreatitis, pancreas cancer and the like; salivary glands diseases such as mumps and the like; renal insufficiency; chronic liver diseases; macroamylasemia; and the like.
The amylase activity has conventionally been measured by various methods and the reagents thereof. The methods can be classified in principle to the following four groups:
(1) Amyloclastic method utilizing starch-iodine reaction. PA0 (2) Saccharogenic method which measures the amount of reducing saccharides formed from starch. PA0 (3) Chromogenic method which measures free chromophores occurred from chromophore-bound starch. PA0 (4) Turbidity process which measures turbidity by starch, especially amylopectin. PA0 (5) a method in which a substrate, i.e. an oligosaccharide having a defined chain length, is cleaved by amylase and then treated by a coupling enzyme such as alpha-glucosidase or maltose phosphorylase to form glucose, and the said glucose is determined by a conventional method (see U.S. Pat. No. 3879263, Japanese Patent Publication (unexamined) Nos. 37096/1978 and 19097/1980, and Japanese Patent Publication (examined) Nos. 33956/1982 and 33957/1982). PA0 (6) a method in which a substrate, i.e. an oligosaccharide having a defined chain length, is cleaved by amylase to form maltose and said maltose is measured by a conventional method (see Japanese Patent Publication (unexamined) No. 80488/1979 and Japanese Patent Publication (examined) No. 27800/1980). PA0 (7) a method in which a substrate, i.e. an oligosaccharide having a defined chain length bonded with p-nitrophenyl group or a substituted aromatic group (aglycone) in the reducing end through glycosidic bond, is cleaved by amylase and then allowed to be contacted to a coupling enzyme, such as glucosidase and the resultant free p-nitrophenyl or aglycone is measured by the absorption in the visible region (Japanese Patent Publication (unexamined) Nos. 25893/1979 and 2199/1985, and Japanese Patent Publication (examined) Nos. 53079/1982, 997/1984 and 13198/1984). PA0 eliminating glucose and/or maltose naturally present in body fluids with a first reagent comprising an enzyme for converting the glucose and/or maltose to glucose-6-phosphate, phosphoglucose isomerase, phosphofructokinase and adenosine-5'-triphosphate, PA0 adding a second reagent comprising a oligosaccharide substrate and a phosphoric acid ester of saccharides and/or saccharic acids to eliminate the phosphoglucose isomerase activity and to convert the oligosaccharide fragments formed by the action of amylase in body fluids to glucose by means of alpha-glucosidase or maltose phosphorylase, and PA0 measuring the amount of the obtained glucose.
These four methods utilize starch or a polymeric amylose or amylopectin as a substrate, but it is difficult to obtain those having uniform quality. Also these methods have ambiguity in the relation between number of cleavage sites by amylase and actually measured values, which is an important defect. In addition, the method (1) indicates inferior analysis accuracy because the measurement is carried out by decrease in absorbance. In the method (2), a boiling treatment is inevitable and the degree of coloring is not constant in each produced cleavage site of oligosaccharide. In the method (3), the reaction period is long and a filtration or centrifuge treatment is necessary. Further, the method (4) is difficult for the substrate to be made uniform and the linear extent of the measured value is narrow.
A method in which amylase activity is measured using an oligosaccharide having a defined chain length as a substrate and a coupling enzyme and a reagent thereof have been recently proposed so as to obtain stoichiometrical relations between the number of cleavage sites by amylase and the measured value. The methods are classified to the following three:
In the method (7), the developed color is unstable because it is changeable by pH, temperature and amount of the co-existent protein and the reproducibility is not so good. The substrate utilized in the method (7) is generally unstable in aqueous solution and it is late in cleaving rate by amylase in comparison with the corresponding oligosaccharide thereto. The method (7) is also complicated in the treatment of an inside standard material which needs to be used in the method (7).
In connection with the improvement of analysis accuracy, it has been found that amylase in body fluids is composed of salivary glands-derived amylase (hereinafter referred to as S) and pancreatic juice-derived amylase (hereinafter referred to as P). A substrate, therefore, is desired to have no difference in its reactivity between both amylases in order to measure the amylase activity in body fluids. In this context, E. Rauscher, U. Neumann, E. Schaich, S. von Buelow, and A. W. Wahlenfeld, Clin. Chem., 31. 14-19 (1985) reports that the oligosaccharide substituted by a substituent at a reducing end has larger P-activity/S-activity ratio than that of the corresponding aligosaccharide substrate. For example, the P-activity/S-activity ratio of p-nitrophenyl maltoheptaose is 1.72 to 1.77, while the P-activity/S-activity of maltoheptaose is 1.45 to 1.50. Maltopentaose has the P-activity/S-activity of 1.0, which is very valuable. Accordingly, in the measurement of the amylase activity in body fluids, the use of the oligosaccharide having a defined chain length as substrate is of value, and an application of it to the methods (5) and (6) is profitable in the clinical examination of amylase activity.
However, the methods (5) and (6) have a tendency to be measured higher than actual value, because glucose (called endogeneous glucose) or maltose (called exogeneous maltose which is recently employed in an intravenous drip) naturally existent in body fluids, especially serum or urine to be clinically examined, are also counted in the measured values according to the methods (5) and (6) wherein glucose and maltose are also produced as reaction intermediates. It is necessary to eliminate them from the body fluids in advance.
For eliminating endogeneous glucose or exogeneous maltose, column methods and enzymatic methods are proposed. In a utilizing the column method, body fluids are applied to a column such as gel filtration, but the column method is not acceptable to daily routine examinations because the operations are complicated.
The enzymatic methods are classified by enzymes being used. For example, Japanese Patent Publication (examined) Nos. 33956/1982 and 33957/1982 disclose that the elimination of endogeneous glucose and exogeneous maltose is carried out by alpha-glucosidase, hexokinase and glucose-6-phosphate dehydrogenase and lactate dehydrogenase. Japanese Patent Publication (unexamined) No. 19097/1980 discloses that oxamic acid makes inactive the above lactate dehydrogenase at the time of measuring amylse activity. Japanese Patent Publication (examined) No. 33958/1982 discloses that endogeneous glucose is removed with hexokinase and the activity of hexokinase is inactivated by an anionic surfactant such as alpha-olefin sulfonate at the time of measuring amylase activity. Further, Japanese Patent Publication (unexamined) No. 203500/1984 discloses that endogeneous glucose is eliminated with a system of mutarotase, glucose oxidase and catalase, and the catalase reaction is then stopped with sodium azide. However, prevention of the reaction to be stopped is not completely done and even high concentration of the above-described substance takes long time to stop the reaction. Also aberration of the measured value is often occurred in these enzymatic elimination methods.
As mentioned above, since the oligosaccharide having a defined chain length is the most suitable substrate for standerizing amylase activity because its cleavage sites are not varied, so much depending on the pancreatic juice-derived amylase and the salivary glands-derived amylase and because the hydrolysis rate is rapid sufficient to be able to continuously follow up the reaction by means of Rate method, a method for measuring amylase activity in combination with an effective elimination method of endogeneous glucose or exogeneous maltose and a reagent thereof is strongly desirable to progress more effectively or constantly the method using the oligosaccharide having a defined chain length.